Studies of P-glycoprotein Drug Interactions - Administrative Supplement for Undergraduate Summer Research

P-糖蛋白药物相互作用的研究 - 本科生暑期研究行政补充

• 批准号: 10810072
• 负责人: INA L URBATSCH
• 金额: $ 0.75万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10810072
• 关键词: ABCB1 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Acceleration; Administrative Supplement; Affinity; Amber; Amino Acids; Antineoplastic Agents; Area; Aromatic Amino Acids; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Availability; Biomedical Engineering; Blood - brain barrier anatomy; Cardiovascular Agents; Cardiovascular system; Cell membrane; Cells; Chemicals; Clinical; Codon Nucleotides; Cryoelectron Microscopy; Cytoplasm; Cytoplasmic Tail; Development; Dimerization; Disease; Documentation; Drug Binding Site; Drug Combinations; Drug Design; Drug Efflux; Drug Interactions; Drug Kinetics; Drug Metabolic Detoxication; Drug Monitoring; Drug Transport; Drug resistance; Drug usage; Energy Transfer; Engineering; Environment; Excretory function; Fluorescence; Fluorescence Spectroscopy; Fluorescent Probes; Funding; Goals; HIV/AIDS; Hydrophobicity; Intestines; Kidney; Kinetics; Knowledge; Laboratories; Learning; Ligand Binding; Lipid Bilayers; Liver; Maps; Measures; Membrane; Mental disorders; Molecular; Molecular Conformation; Monitor; Multi-Drug Resistance; Nifedipine; Nucleotides; Paclitaxel; Pharmaceutical Preparations; Positioning Attribute; Prazosin; Property; Pump; Recombinant Proteins; Rhodamine 123; Roentgen Rays; Scientist; Screening procedure; Site; Structure; Surface; System; Technology; Terminator Codon; Tertiary Protein Structure; Testing; Therapeutic; Toxin; Transmembrane Domain; Tryptophan; United States Food and Drug Administration; Variant; Vinblastine; X-Ray Crystallography; absorption; biophysical techniques; cancer drug resistance; cancer therapy; clinically relevant; combat; efflux pump; experience; experimental study; functional group; inhibitor; insight; molecular pump; nanodisk; novel strategies; novel therapeutics; prevent; protein purification; protein reconstitution; rational design; reconstitution; single molecule; success; summer research; tool; tryptophan analog; undergraduate student; uptake

PROJECT SUMMARY/ABSTRACT (of the original funded project) P-glycoprotein (Pgp) is a molecular pump that detoxifies cells by transporting hundreds of structurally unrelated toxins out of the cell. Pgp limits uptake in the intestines, and enhances excretion of drugs in the liver, kidney and blood-brain barrier, of many drugs that are used for treatment of cancers, HIV/AIDS, psychiatric illnesses, and cardiovascular conditions. It is among the seven most important transporters responsible for regulating drug absorption and disposition that now require documentation of drug interactions for approval of any new drugs by the US Food and Drug Administration (FDA). Pgp is an ATP-binding cassette transporter with two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). It uses ATP hydrolysis to pump substrates across the cell membranes. Our recent X-ray structures of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis that therapeutic drugs bind to different subsets of residues within defined subpockets in the TMDs of the protein. Our general approach is to introduce tryptophans (Trps) at strategic positions in order to monitor drug binding. The Trps will be introduced on the background of a new fully functional Trp-less Pgp, or a low-Trp Pgp that retains three native conformationally sensitive Trps in the cytoplasmic domains. Using fluorescence changes, such as quenching, and resonance energy transfer (FRET), we will map out sites of interaction of the purified protein with prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common therapeutic drugs and newly identified inhibitors. We will further insert a fluorescent Trp analog (L-Anap) into wild-type Pgp using the amber stop codon suppression strategy to explore monitoring drug binding in biological cell membranes. By determining how drugs and inhibitors modulate the cooperativity and conformational dynamics of this multidomain transporter, we will gain unique insight into the mechanisms of drug binding and their effects on Pgp function. With these new approaches, we will address the molecular mechanism and kinetics of drug/inhibitor binding, determine synergistic effects, and refine the mechanisms of drug-drug interactions in Pgp. The information will pave the way to new analytical approaches to refine Pgp drug interaction studies of old and new drugs, and will be invaluable to redesign drugs with clinically favorable pharmacokinetics and accelerate pharmacotherapeutic developments.

项目概要/摘要（原始资助项目） P-糖蛋白（Pgp）是一种分子泵，通过转运数百种结构上无关的 把毒素从细胞中排出Pgp限制了肠道的吸收，并增强了药物在肝、肾和 血脑屏障，许多药物用于治疗癌症，艾滋病毒/艾滋病，精神疾病， 心血管疾病它是负责调节药物的七种最重要的转运蛋白之一 吸收和处置，现在需要记录药物相互作用，以批准任何新药， 美国食品和药物管理局（FDA）。Pgp是一种ATP结合盒转运蛋白， 跨膜结构域（TMD）和两个核苷酸结合结构域（NBD）。它利用ATP水解作用 底物穿过细胞膜。我们最近的Pgp X射线结构鉴定出疏水性和芳香性 有助于不同抑制剂与药物结合位点结合的氨基酸。在本提案中，我们将测试 治疗药物与TMD中定义的亚口袋内的不同残基子集结合的假设 蛋白质。我们的一般方法是在战略位置引入Trps，以监测 药物结合Trps将在新的全功能Trp-少Pgp或低Trp的背景下引入。 在胞质结构域中保留三个天然构象敏感Trps的Pgp。使用荧光 变化，如淬灭和共振能量转移（FRET），我们将绘制出相互作用的网站， 纯化的蛋白质，其具有占据生物化学定义的和不同的结合位点的原型底物，以及 与普通治疗药物和新鉴定的抑制剂的那些相同。我们将进一步插入荧光色氨酸 类似物（L-Anap）转化为野生型Pgp，采用琥珀终止密码子抑制策略探索监测药物 结合在生物细胞膜上。通过确定药物和抑制剂如何调节协同性， 这种多结构域转运蛋白的构象动力学，我们将获得独特的洞察药物的机制， 结合及其对Pgp功能的影响。通过这些新方法，我们将解决分子机制 和药物/抑制剂结合的动力学，确定协同效应，并完善药物-药物结合的机制。 Pgp的相互作用这些信息将为改进Pgp药物相互作用的新分析方法铺平道路 新旧药物的研究，对于重新设计具有临床有利药代动力学的药物将是无价的 加速制药业的发展。